This application is the U.S. National phase under 35 U.S.C. xc2xa7 371 of International Application PCT/EP98/00493, filed Jun. 26, 1998, which claims priority of Australia application AU PO 7593, filed Jun. 26, 1997.
This invention relates to plant and viral promoters that confer high level gene expression on transgenic plants harbouring the promoters. The invention also relates to utilisation of the promoters in the construction of recombinant genes for plant transformation to enable expression at a certain time in a certain tissue and at a certain rate. In particular, the invention relates to promoters isolated from different isolates of banana-infecting badnaviruses.
Genetic engineering of plants has proven to be an alternative method for plant breeding and for the introduction of new desirable traits that are reflected in altered phenotypes. In addition, it provides a valuable tool for biological research. Plant genetic manipulation focuses on the cellular level of organisation and involves the interfacing of all aspects of cell biology, molecular biology and gene transfer procedures (Sharp et al., Food Technology, February 1984, pp. 112-119). The genetic engineering tools of tissue culture, somaclonal and gametoclonal variation, cellular selection procedures and recombinant DNA are either indirectly or directly concerned with the enhanced expression and transfer of genes. An essential problem for this is the choice of a suitable promoter that results in the desired rate, location and time of gene expression. In the majority of applications of plant genetic engineering, a strong promoter is required to ensure that a sufficient amount of gene product is expressed. These applications include genetic manipulation of plants to obtain disease resistance or tolerance against plant-infecting viruses, bacteria, fungi or nematodes, to obtain resistance against herbivores, to obtain resistance against herbicides, heavy metals and selectable marker reagents, to obtain resistance against abiotic factors (e.g., draught, salt, cold, and anaerobic conditions) to conduct functional analyses of genes and gene products for research, to confer silencing or enhancement of genes and gene products (modulation of gene expression), to modify the composition of macromolecules and secondary metabolites (e.g., to increase nutritional value or to alter structural composition), to modify plant development, and to improve fruit or crop quality. (e.g., post harvest shelf life or disease resistance).
The function and mode of action of promoters have been studied extensively in both monocot and dicot plants. In most cases reporter genes such as the uidA gene encoding for xcex2-glucuronidase (GUS: Jefferson et al., EMBO J. 6, 3901-3907 [1987]) or genes encoding anthocyanin production or the jellyfish green fluorescent protein (GFP; Chalfie et al., Science 263, 802-805 [1994]) are used to assay promoter activity in transient or stable gene expression systems. Promoters derived from monocot species often do not exhibit a regulated pattern of expression in transgenic dicots, whereas in transgenic monocots, they show a highly regulated expression (Shimamoto, Current Opinion in Biotechnology, 5, 158-162 [1994]). Highly regulated expression patterns have been demonstrated for several promoters in transgenic monocots, even though there is no absolute specificity. These include light-inducible and leaf-specific promoters, seed-specific promoters, meristem-specific promoters, root-specific promoters, flower-specific promoters, hormone-inducible promoters, pathogen-inducible promoters and constitutive promoters. Within monocot and dicot plants it has been shove that promoters derived from species of the same group reveal the same or similar highly regulated expression pattern (Shimamoto, 1994, supra).
For many purposes in plant genetic engineering, a strong nearly constitutive promoter is required to ensure sufficient expression throughout the plant. Several strong nearly constitutive promoters for the genetic manipulation of plants have been patented (e.g., the 35S promoter of cauliflower mosaic virusxe2x80x94see U.S. Pat. No. 5,352,605, U.S. Pat. No. 5,164,316, U.S. Pat. No. 5,196,525. U.S. Pat. No. 5,322,938 and U.S. Pat. No. 5,359,142). However, having more than one nearly constitutive promoter can be very useful when several different genes need to be expressed in plants (gene pyramiding). It has been frequently observed that gene silencing occurs in plants transformed with several genes that are each regulated by the same promoter (Flavell, Proc. Natl. Acad. Sci. USA 91, 3490-3496 [1994]; Finnegan and McElroy, Bio/Technology 12, 883-888 [1994]; Matzke et al., Mol. Gen. Genet. 244, 219-229 [1994]: Park et al., The Plant Journal 9, 183-194 [1996]). This problem is thought to be caused by homology-based genetic interference and can be avoided using different promoters for gene pyramiding.
It is an object of the present invention to provide promoters operative in plant cells which can be used in genetic engineering for regulation of gene expression.
It is a further object of the invention to provide at least part of a chimaeric gene comprising one or more of the described promoters operatively linked to DNA encoding an RNA and/or polypeptide.
According to a first embodiment of the invention, there is provided a promoter operative in a plant cell, said promoter comprising:
1) isolated DNA from a badnavirus having a sequence as defined by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;
2) isolated DNA which is a viral homologue or a plant genome-derived variant of the DNA of (1).
3) a promoter-active portion of the isolated DNA of (1) or (2);
4) an isolated DNA which hybridises under stringent conditions to the DNA of (1) or (2); or
5) a promoter-active portion of the isolated DNA of (4).
According to a second embodiment of the invention, there is provided a DNA construct comprising at least one gene having at least one promoter according to the first embodiment operatively linked to a coding sequence.
According to a third embodiment of the invention, there is provided a DNA construct comprising:
1) a first gene having at least one promoter according to the first embodiment operatively linked to a coding sequence; and
2) a second gene having a promoter operatively linked to a coding sequence, wherein the expression product of said second gene coding sequence modulates activity of the expression product of said first gene coding sequence.
According to a fourth embodiment of the invention, there is provided a method of expressing a product in a plant cell, said method comprising introducing a DNA construct according to the second embodiment or an RNA transcript of said construct into cells of a plant, wherein said DNA construct or RNA transcript coding sequence encodes said product.
According to a fifth embodiment of the invention, there is provided a plant cell, wherein the genome of said plant cell includes a DNA construct according to the second embodiment or the third embodiment.
According to a sixth embodiment of the invention, there is provided a plant, plant tissue or reproductive material of a plant, wherein said plant, plant tissue or reproductive material comprises cells according to the fifth embodiment.